Reversed endcapping and bonding of chromatographic stationary phases using hydrosilanes

ABSTRACT

A process for producing a chromatographic stationary phase for use in reversed-phase chromatography. Chromatographic stationary phases prepared according to the methods of the current invention and liquid chromatography columns, which include the stationary phases, are also provided.

FIELD OF THE INVENTION

The present invention relates to chromatographic stationary phases foruse in liquid chromatography. More particularly, the present inventionrelates to chromatographic stationary phases for use in reversed-phaseHPLC.

BACKGROUND

Silica particles are, by far, the most widely used supports forreversed-phase liquid chromatography stationary phases. The highmechanical stability, monodisperse particles, high surface area, andeasily tailored pore size distributions make silica superior to othersupports in terms of efficiency, rigidity, and performance. Silicabonding chemistry also allows for a wide variety of stationary phaseswith different selectivities to be made on silica.

Silanes are the most commonly used surface modifying reagents in liquidchromatography. For example, An Introduction to Modern LiquidChromatography, Chapter 7, John Wiley & Sons, New York, N.Y. 1979; J.Chromatogr. 352, 199 (1986); J. Chromatogr. 267, 39 (1983); and Advancesin Colloid and Interface Science 6, 95 (1976) each disclose varioussilicon-containing surface modifying reagents.

Typical silane coupling agents used for silica derivatization have thegeneral formula EtOSiR₁R₂R₃ or ClSiR₁R₂R₃, where each R representsorganic groups, which can differ from each other or all be the same. Forreversed-phase chromatography, the silane coupling agent hastraditionally been

—Si(CH₃)₂(C₁₈H₃₇), where C₁₈H₃₇, an octadecyl group, yields ahydrophobic surface. The reaction, when carried out on the hydroxylatedsilica, which typically has a maximum surface silanol concentration ofapproximately 8 μmol/m², does not go to completion due to the stericcongestion imposed by the octadecyl groups on the coupling agent.

To improve the quality of the original chemically bonded phase byblocking access to some residual silanol groups on the silica surface,the bonded phase is usually further endcapped using small organicsilanes. The endcapping is usually carried out with compounds able togenerate trimethylsilyl groups, (CH₃)₃—Si—, the most popular beingtrimethylchlorosilane (TMCS) and hexamethyldisilazane (HMDS). Themajority of free surface silanols, which are under thedimethyloctadecylsilyl groups, cannot react with the endcapping reagentsbecause of steric hindrance. In the traditional endcapping step, only˜0.2 μmol/m² surface silanol groups are bonded based on the carbonloading data. The highest coverage attained in laboratory studies hasbeen ˜4.5 μmol/m², while the coverage available in commercialchromatography columns is much less, usually on the order of 2.7-3.5μmol/m², even after endcapping.

These residual surface silanols interact with basic and acidic analytesvia ion exchange, hydrogen bonding and dipole/dipole mechanisms.However, this secondary interaction between analytes and residualsilanol groups results in increased retention times, excessive peaktailing, especially at mid pH range for basic compounds, andirreversible adsorption of some analytes.

To overcome the problems of residual silanol activity, many methods havebeen tried such as the use of ultrapure silica, the use of carbonizedsilica, the coating of the silica surface with polymeric compositions,the endcapping of residual silanol groups, and the addition ofsuppressors such as long chain amines to the eluent. In practice,however, none of these approaches has been totally satisfactory. Ageneral review of silica support deactivation is given by Stella et al.[Chromatographia (2001), 53, S-113-S115].

A method to eliminate surface silanols by extreme endcapping isdescribed in U.S. Pat. No. 5,134,110. While traditional endcapping canphysically bond some residual silanol groups, at least 50% of thesurface silanols remain unreacted. U.S. Pat. No. 5,134,110 describes anendcapping method for octadecyl-silylated silica gel by high temperaturesilylation. Polymeric chemically bonded phases originated fromtrichlorosilanes were endcapped using hexamethyldisilazane orhexamethylcyclo-trisiloxane at very high temperature, above 250° C., ina sealed ampoule. The resulting endcapped phases were shown to performwith excellence on the Engelhardt test. This result was explained by theformation of dimethylsilyl loop structures on the surface, leading tothe elimination of silanols. This method had the disadvantage in that itwas used on a polymeric phase, and polymeric phases usually have poormass transfer and poor reproducibility. Also the high temperature ofsilylation in a sealed ampoule is not practical and difficult to performcommercially, as compared with the traditional liquid phase endcappingprocedure.

Another method of reducing the effect of surface silanols is tointroduce polar embedded groups in the octadecyl chain. These embeddedgroups, generally contain nitrogen atoms and amides such as disclosed inEuropean Patent No. EP0397301 and carbamates such as disclosed in U.S.Pat. No. 5,374,755. Most recently, urea groups have been shown to reducethe undesirable silanol interactions. Phases with incorporated polargroups clearly exhibit lower tailing factors for basic compounds, whencompared with traditional C-18 phases. Some mechanisms have beenproposed, while some evidence leads to the belief that the surface layerof an embedded polar group phase should have a higher concentration ofwater due to the hydrogen bonding ability of the polar groups near thesilica surface. This virtual water layer suppresses the interaction ofbasic analytes with residual surface silanols and permits separationwith mobile phase having 100% water.

A disadvantage of this approach is that the presence of the water layerseems to contribute to a higher dissolution rate of the silica support,as compared to their alkyl C-8 and C-18 counterparts. In a systematiccolumn stability evaluation, an embedded amide polar stationary phasewas shown to be less stable. This result may be predictable, due to thehigher water content near the underlying silica surface for polarembedded phases. The embedded polar groups also cause adsorption of someanalytes when the phases are hydrolyzed or the phases are not fullyreacted during phase preparation, leaving amine or hydroxyl groups onthe surface. For example, the hydrolyzed amide phase leaves aminopropylmoieties on the surface, that strongly adsorb acidic and polarcompounds, causing their peaks to be tailed, or to be missingcompletely.

The polar embedded phases are also more hydrophilic than the traditionalC-18 phases, enhancing the retention of polar compounds, whereas theretention of hydrophobic analytes is much less on polar-embedded columnsthan on the traditional C-18 columns. As a result, the phase selectivityis quite different from traditional C-18, which causes a change in theorder in which analytes elute relative to each other from the column.Consequently, methods developed on traditional C-18 columns cannot betransferred to polar embedded phase columns.

Another method for reducing the effect of surface silanols is to use aphase that can sterically protect surface silanols. U.S. Pat. No.4,705,725 to Du Pont discloses bulky diisobutyl (with C-18) ordiisopropyl (with C-8, C3, phenyl propyl, cyano propyl) side chaingroups (Zorbax® Stable Bond reversed-phase columns) that stabilize bothlong and short chain monofunctional ligands and protect them fromhydrolysis and loss at low pH. The bulky side groups increase thehydrolytic stability of the phase. Such a moiety is less vulnerable todestruction at low pH, and better shields the underlying silanols. Thesterically protected phases are extremely stable at low pH. Thesterically protected silane phases are not endcapped; therefore, theloss of small, easily hydrolyzed endcapping reagents under acidic mobilephase condition is avoided. At pH<3, the phase has excellentperformance, in terms of peak shape, retention, reproducibility, andlifetime. In this pH range, the silanol groups on a type B silica arenearly completely protonated, and as a result, they do not act as sitesfor secondary interaction. The coverage density is, however, much lowerthan for dimethyl ODS phases. The ligand density of diisobutyloctadecylphase is ˜2 μmol/m² when compared to the conventional dimethyloctadecylphase with a ligand density of 3.37 mmol/m².

U.S. Pat. No. 5,948,531 discloses the use of bridged propylene bidentatesilanes or a bidentate C-18 phase (Zorbax® Extend-C-18 columns), torestrict analyte access to residual silanols by incorporating apropylene bridge between two C-18 ligands. The bidentate C-18 phaseretains the benefits of monofunctional silane phases (high columnefficiency, reaction repeatability) while demonstrating good stabilityin high and low pH mobile phases. Zorbax Stable-Bond C-18 (SB-C-18) andZorbax Extend-C-18 columns also have very similar selectivity to thetraditional C-18 columns.

Basic compounds appear in widely divergent areas, such as theenvironmental, chemical, food, and pharmaceutical industries. In thelatter, in particular, over 80% of commercialized drugs are estimated topossess a basic function. Therefore, it is of crucial importance todevelop practical HPLC stationary phases having minimized surfacesilanol activity.

SUMMARY OF THE INVENTION

The present invention is directed a process for producing achromatographic stationary phase for use in reversed-phasechromatography by providing an inorganic oxide support materialcomprising surface hydroxyl groups; reacting the surface hydroxyl groupswith at least one endcapping reagent having a formula selected from I,II, and III:

wherein, R¹ is hydrogen or methyl, X is halogen, OR² or NR³R⁴, and Z is—NH— or —NR⁵—, wherein R² is C₁ to C₃₀ alkyl, R³ and R⁴ areindependently hydrogen or C₁ to C₃₀ alkyl, and R⁵ is methyl or ethyl, toprovide a modified particulate support material having at least oneendcap Si—H group; converting at least one endcap Si—H group to anendcap silanol group; and reacting the endcap silanol group with atleast one silane coupling agent selected from IV, V, VI, and VII:

wherein R⁶⁻¹⁵ are independently hydrogen or C₁ to C₃₀ hydrocarbyl,optionally substituted with at least one reversed-phase chromatographyligand; Y is halogen, OR² or NR³R⁴, wherein R² is C₁ to C₃₀ alkyl, andR³ and R⁴ are independently hydrogen or C₁ to C₃₀ alkyl; m is 1 orgreater; and n is 1 or greater to provide a functionalized particulatesupport material.

Chromatographic stationary phases prepared according to the methods ofthe current invention and liquid chromatography columns, which includethe stationary phases, are also provided.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a novel improvement to a chromatographicstationary phase for use in reversed-phase chromatography. It has beenfound that reversing endcapping and bonding by first endcapping thesurface hydroxyl groups of an inorganic oxide support material withsmaller mono and dimethyl hydrosilanes, instead of the traditionaltrimethyl silanes, followed by converting at least one endcap Si—H groupto an endcap silanol group and reacting the endcap silanol group with atleast one silane coupling agent, results in improved total coverage ofsurface silanols, which surface coverage reaches as high as 5-7 μmol/m².For example, silica bonded with —SiMe₂H has surface coverage of 5.10μmol/m², and silica bonded with difunctionalbis(dimethylamino)methylsilane has surface coverage of 6.9 μmol/m².

A more hydrophobic inorganic oxide surface that is more resistant todissolution at middle and high pH conditions is also produced. Theendcap silanol groups on the functionalized silica support material aremuch less acidic than silica surface silanols. Furthermore, thefunctionalized support material provides exceptional peak shapes forstrong bases.

According to the process of the current invention, the endcapping monoand/or dimethyl hydrosilanes are introduced to the chromatographicsupport prior to the introduction of the hydrophobic phase. Suitableinorganic oxide support materials include those typically utilized inliquid chromatography, for example, silica, hybrid silica, an example ofwhich is disclosed in U.S. Pat. No. 4,017,528, the contents of which areincorporated herein by reference, alumina, titanium oxide, and zirconiumoxide. Furthermore, the support can be in any form suitable for use inliquid chromatography. Suitable forms include porous particles,non-porous particles, porous membranes, and porous monoliths, an exampleof which is disclosed in U.S. Pat. No. 6,210,570 the contents of whichare incorporated herein by reference. As used herein, the term “porous”means any chromatographically-suitable degree of porosity. The term“porous particles” also includes superficially porous particles, forexample, non-porous particles coated with a porous outer layer.

The endcapping is done in an inert solvent, such as toluene,tetrahydrofuran or another inert hydrocarbon, under reflux conditionsaccording to methods that are well known in the art. The endcappingagent may be introduced using any silane capable of generating a mono ordimethyl hydrosilyl groups in solution at reflux or in a gas phasereaction since small mono or dimethyl hydrosilanes have low boilingpoint temperatures.

Preferred endcapping reagents according to the current invention areselected from formulas I, II, and III:

wherein R⁶⁻¹⁵ are independently hydrogen or C₁ to C₃₀ hydrocarbyl,optionally substituted with at least one reversed-phase chromatographyligand; Y is halogen, OR² or NR³R⁴, wherein R² is C₁ to C₃₀ alkyl, andR³ and R⁴ are independently hydrogen or C₁ to C₃₀ alkyl; m is 1 orgreater; and n is 1 or greater. Preferably, at least one of R⁶⁻¹⁵ is asaturated or unsaturated hydrocarbyl, a cyclic hydrocarbyl, or an arylhydrocarbyl. More preferable, at least one of R⁶⁻¹⁵ is C₁₈H₃₇ or C₈H₁₇.Where Y is a halogen, an acid scavenger, such as imidazole or pyridineis added to the reaction mixture. Typical methodologies for introducingthe hydrophobic phase are described in Silane Coupling Agents:Connecting Across Boundaries, published by Gelest, Inc. (2004), andavailable at www.gelest.com/company/pdfs/couplingagents.pdf. The methodsdescribed therein are also useful for introducing the endcapping agent.The current invention does not depend on the manner in which the silanecoupling agent is introduced, and it is contemplated that the currentinvention will be applicable to all conventionally known ways ofintroducing the silane coupling agent.

The silane coupling agents used for the hydrophobic phase may containany ligand commonly used in stationary phases for reversed-phase HPLC.For example, when bonded to endcap silanol groups, the resultingstructure of formula IV is:

wherein the oxygen atom forms the bond to the endcap agent and R⁶⁻⁸ aredefined as above. As used herein, the term hydrocarbyl means any ligandcomprising a straight chain, branched, or cyclic carbon backbone.Further, the ligand may contain one or more unsaturated moieties and inthe case of cyclic moieties, may be aryl. Still further, the ligand maybe substituted with any moiety commonly used in reversed-phase HPLC.Exemplary ligands used for stationary phases for reversed-phase HPLCinclude alkyl, aryl, cyano, diol, nitro, amides, amines, phenyl,nitrites, butyl, octyl, octadecyl, cation or anion exchange groups, andembedded polar functionalities, for example, amides, ethers, andcarbamates. For the purposes of the current invention the identity ofthe specific coupling agent is not critical, as the invention isapplicable generally to reversed-phase chromatographic stationaryphases.

Chromatographic stationary phases prepared according to the methods ofthe current invention and liquid chromatography columns, which includethe stationary phases, are also provided. The chromatographic stationaryphase can include an inorganic oxide support material having bondedthereto, via Si—O bonds, at least one silane of formula VIII:

wherein, R¹ is hydrogen or methyl, X is halogen, OR² or NR³R⁴, and Z is—NH— or —NR⁵—, wherein R² is C₁ to C₃₀ alkyl, R³ and R⁴ areindependently hydrogen or C₁ to C₃₀ alkyl, and R⁵ is methyl or ethyl. Inthe case that an endcapping agent according to formula III is used, theendcapping agent is capable of producing two Si—O bonds, and thus,occupies two surface hydroxyl. Where X is a halogen, an acid scavenger,such as imidazole or pyridine is added to the reaction mixture. Whenbonded to the inorganic oxide support material, the endcapping agentshave formulas selected from IX and X:

wherein the oxygen atoms form the bond to the support material.

Preferably, Si—H groups are converted to Si—OH in a dioxiran solution oran Oxone™ (active ingredient: potassium peroxymonosulfate, KHSO₅ [CAS-RN10058-23-8]) buffer solution.

The silane coupling agent used to create the hydrophobic phase may beintroduced in any manner commonly known in the art. Typically, thehydrophobic phase is introduced by reacting the endcap Si—OH groups witha silane selected from formulas IV, V, VI, and VII:

wherein R¹ and R^(1′) are independently hydrogen or methyl and R⁸, R⁹and R¹⁰ are independently hydrogen or C₁ to C₃₀ hydrocarbyl, optionallysubstituted with at least one reversed-phase chromatography ligand.

EXAMPLES Example 1 Endcapping Surface Silanols of a Silica Support withSiMe₂H

Surface silanols of a silica support were endcapped with SiMe₂Haccording to the following method: 32.56 g silica with surface area of260 m²/g was charged into a 250 ml three necked flask equipped with aBarrett trap and a water condenser. 150 ml toluene was added. 30 mltoluene was distilled out and collected in the Barrett trap. After themixture was allowed to cool below 100° C., the Barrett trap was removed,and a new water condenser was attached. (Dimethylamino)dimethylsilane(8.37 g, 81.26 mmol) was added. The mixture was then stirred underreflux conditions overnight (18-24 hours). The silica was filtered,washed with 50 ml toluene, 50 ml THF, and 50 ml CH₃CN, and air-dried.The silica was then dried under vacuum at 120° C. overnight. The silicahad a carbon loading of 3.16%, 5.10 μm/m² coverage.

Example 2 Endcapping Surface Silanols of a Silica Support with SiMeH

Surface silanols of a silica support were endcapped with SiMeH accordingto the following method: 38.18 g silica with surface area of 260 m²/gwas charged into a 250 ml three necked flask equipped with a Barretttrap and a water condenser. 150 ml toluene was added. 30 ml toluene wasdistilled out and collected in the Barrett trap. After the mixture wasallowed to cool below 100° C., the Barrett trap was removed, and a newwater condenser was attached. Bis(dimethylamino)methylsilane (8.37 g,81.26 mmol) was added. The mixture was then stirred under refluxconditions overnight (18-24 hours). The silica was filtered, washed with50 ml toluene, 50 ml THF. The silica was reslurried in 120 ml THF/water(80/20 v/v), and was stirred under reflux conditions for 1 hour. Thesilica was filtered, washed with 50 ml toluene, 50 ml THF and 50 mlCH₃CN, and air-dried. The silica was then dried under vacuum at 120° C.overnight. The silica had a carbon loading of 2.14%, 6.90 μm/m²coverage.

Example 3 Converting Si—H to Si—OH

A dioxirane solution was used to convert Si—H groups to Si—OH. Thesynthesis of dioxirane is described in J. Org. Chem., Vol. 50, No. 16,2847-2853 (1985). A dioxirane solution was prepared as follows: amixture of 20 ml water/15 ml acetone was added to a mixture of 20 mlwater/15 ml acetone/24 g NaHCO₃ in a three necked flask equipped with anadditional funnel, an additional funnel for solid, and a connectorconnected to a dry ice condenser with a cold trap below. At the sametime, 50 g Oxone™ (active ingredient: potassium peroxymonosulfate, KHSO₅[CAS-RN 10058-23-8]) was added. After addition, the mixture was stirredfor 15 minutes. The additional funnels were removed. N₂ was purged whileslight vacuum was applied. The product dioxirane was condensed at thedry ice condenser and collected in the flask in the cold trap (about 30ml).

Endcapped silica prepared according to Example 1 (4.6 g silica particlesbonded with —SiMe₂H) was then added into the resulting dioxiranesolution in the flask. The mixture was stirred at room temperature for 2hours. The silica was filtered, washed with acetone, and dried undervacuum.

In an alternative process, endcapped silica was reacted with an Oxone™buffer solution and acetone to convert Si—H to Si—OH. The Oxone™ buffersolution was prepared as follows: 50 g Oxone™ was added into 400 ml 20mM K₂HPO₄ solution cooled in a ice-water bath. 1M KOH solution was addeduntil pH reached between 7.0-7.5.

The Oxone™ buffer solution was reacted with endcapped silica preparedaccording to Example 1 (silica bonded with —SiMeH (carbon loading2.14%)) as following: 7.21 g silica with —SiMeH surface was added to 60ml acetone cooled in an ice-water bath. The neutral Oxone™ buffersolution prepared above was then added dropwise while the mixture waswell stirred. After addition, the mixture was stirred for 3 hours, thenfiltered, washed with 150 ml water/acetone (2:1 v/v ratio) and 50 mlwater/acetone (1:1 v/v ratio). The silica was reslurried in 100 ml HClsolution (pH 2.0)/acetone (1:1 v/v ratio) for few minutes, filtered,washed with 50 ml water/acetone (1:1 v/v ratio), 30 ml acetone and 30 mlACN, and then was dried under vacuum at 110° C. overnight. The silicahas a carbon loading of 1.73%.

Example 4 Reacting Endcapped Silanol with Coupling Agent

A coupling agent, e.g. a C18 silane, was bonded the endcapped silica ofExample 2. 5.30 g silica with surface SiMeH converted to SiMe-OH (carbonloading 1.73%) was charged into a 250 ml three necked flask equippedwith a Barrett trap with a water condenser on it. 50 ml toluene wasadded. 30 ml toluene was distilled out and collected in the Barretttrap. After the mixture was allowed to cool below 100° C., the Barretttrap was removed, and a new water condenser was attached.(Dimethylamino)dimethyloctadecylsilane (31.92 g, 89.74 mmol) was added.

The mixture was then stirred under reflux conditions overnight (18-24hours). The silica was filtered, washed with 50 ml toluene, 50 ml THF,and 50 ml CH₃CN, and air-dried. The silica was then dried under vacuumat 120° C. overnight. The silica has a carbon loading of 12.49%. Thesilica was then further endcapped with trimethylsilane as usual, with afinal carbon loading of 12.58%.

The present invention has thus been described with reference to specificnon-limiting examples. The full scope of the present invention will beapparent from the appended claims.

1. A process for producing a chromatographic stationary phase for use inreversed-phase chromatography, comprising: providing an inorganic oxidesupport material comprising surface hydroxyl groups; reacting thesurface hydroxyl groups with at least one endcapping reagent having aformula selected from the group consisting of formulas I, II, and III:

wherein, R¹ is hydrogen or methyl, X is halogen, OR² or NR³R⁴, and Z is—NH— or —NR⁵—, wherein R² is C₁ to C₃₀ alkyl, R³ and R⁴ areindependently hydrogen or C₁ to C₃₀ alkyl, and R⁵ is methyl or ethyl, toprovide a modified particulate support material having at least oneendcap Si—H group; converting at least one endcap Si—H group to anendcap silanol group; and reacting the endcap silanol group with atleast one silane coupling agent selected from the group consisting offormulas IV, V, VI, and VII:

wherein R⁶⁻¹⁵ are independently hydrogen or C₁ to C₃₀ hydrocarbyl,optionally substituted with at least one reversed-phase chromatographyligand; Y is halogen, OR² or NR³R⁴, wherein R² is C₁ to C₃₀ alkyl, andR³ and R⁴ are independently hydrogen or C₁ to C₃₀ alkyl; m is 1 orgreater; and n is 1 or greater to provide a functionalized particulatesupport material.
 2. The process according to claim 1, wherein theend-capping reagent is a silane according to formula I or II, and Y is ahalogen, and the process further comprising adding an acid scavenger tothe inert solvent.
 3. The process according to claim 1, wherein at leastone of R⁶⁻¹⁵ is substituted with at least one reversed-phasechromatography ligand.
 4. The process according to claim 1, wherein atleast one of R⁶⁻¹⁵ is a saturated or unsaturated hydrocarbyl, a cyclichydrocarbyl, or an aryl hydrocarbyl.
 5. The process according to claim4, wherein said hydrocarbyl is C₁₈H₃₇ or C₈H₁₇.
 6. The process accordingto claim 1, wherein said inorganic oxide support material is selectedfrom the group consisting of porous particles, non-porous particles,porous membranes, and porous monoliths.
 7. The process according toclaim 1, wherein said inorganic oxide support material is selected fromthe group consisting of silica, hybrid silica, alumina, titanium oxide,and zirconium oxide.